TrxT and dhd are dispensable for Drosophila brain development but essential for l(3)mbt brain tumour growth

Expression of the Drosophila cancer-germline (CG), X-linked, head-to-head gene pair TrxT and dhd is normally germline-specific but becomes upregulated in brain tumours caused by mutation in l(3)mbt. Here, we show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable, taking into account that these two genes are never expressed together under normal conditions. We also show that TrxT, but not dhd, is crucial for the growth of l(3)mbt allografts, hence suggesting that the initial stages of tumour development and long-term tumour growth may depend on different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among CG genes that map to the X chromosome. Our results identify a first example of an X-linked, head-to-head CG gene pair in Drosophila, underpinning the potential of such CG genes, dispensable for normal development and homoeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.

The manuscript by Molnar and colleagues is a follow-up from the authors' previously published work (Molnar et al 2019), where they demonstrated differential sex-linked gene expression in Drosophila l(3)mbt brain tumours and identified the thioredoxin proteins TrxT and Dhd in the mbt tumour signature (MBTS).In the current manuscript, the authors generated genetic mutants for both genes using CRISPR TrxTKO and dhdKO and a deficiency that spans both genes (Df(1)J5) to understand the role played by these thioredoxins in normal larval brain development and l(3)mbt tumour development.Both genes were found to be largely dispensable for normal larval brain development, however the authors uncovered a role in l(3)mbt tumour growth and development.Interestingly, although both TrxT and Dhd were required for l(3)mbt tumour development, they appeared to have distinct roles in longterm tumour growth, as assessed by allograft-induced lethality.Furthermore, the authors also investigated the link between the sexually dimorphic signatures of l(3)mbt tumours and the thioredoxins and suggest that both genes are required for the differential gene expression observed between the sexes.Some of the conclusions are fairly well supported by the data presented.However, there are some aspects that are potentially not fully explored and that would provide more weight to the claims made by the authors.Some of these can be addressed by clarifications made in the text and/or figures and others would benefit from additional immunofluorescence staining experiments.Specific comments are provided below.
1. Figures should include information regarding the sex of the larvae, particularly as there has been a previously reported sex-linked effect in the phenotypes analysed.(e.g. in Figure 2 and Figure S1, where Indication of the sex of the animals should be provided in the figure and not just in the figure legend).
2. Data regarding fertility.Can this be shown in a table format?Are dhdKO females fully sterile?What are the fertility levels of Df(1)J5? 3. Are dhd and TrxT the only genes affected by Df(1)J5?Is there transcriptional data from Df(1)J5 animals to suggest that nearby genes are not affected by the deficiency?Of particular interest would be to assess if snf is affected or not as it is a known regulator of gene expression and splicing.4. In Figure 1C, statistical test plus indication of significance is not presented.5. Related to Figure 1D.Additional neural markers could be assessed in dhdKO and TrxTKO flies.Whilst the gross morphology of the brain does not seem to be affected, there is a possibility that cell specification is affected.Specific markers for the NE, MED and CB could be used to assess this in more detail, particularly as the DE-cad images shown for dhdKO and TrxTKO flies seem to differ slightly from the control.
6. Related to Figure 2A, images from TrxTKO; l(3)mbtts1, dhdKO and l(3)mbtts1 should be added at the very least in a supplementary figure.Additionally, data for NE/BL ratio should be provided for dhdKO, TrxTKO and Df(1)J5 in the absence of l(3)mbtts1 tumours.Related to Figure S1, quantification of NE/BL ratio for female lobes should be added to the figure .7. Related to Figure 2B and Figure S1, three rows of images are presented for each genotype.It is unclear whether these correspond to brain lobes from different larvae or different confocal planes from the same animal.This should be clarified in the figure and/or figure legend.Related to this, in addition to the anti-DE-cadherin data, it would be informative to include immunofluorescence data using antibodies such as anti-Dachshund (lamina), anti-Elav (medulla cortex) and anti-Prospero (central brain and boundary between central brain and medulla cortex) (as assessed in e.g.Zhou and Luo, J Neurosci 2013) in the mbt tumour situation to accurately describe regions disrupted by the tumours.
8. Authors should clarify how the NE was defined when mbt tumours are generated, as it is severely affected.From the images provided, it is unclear which region corresponds to NE or how the NE/BL ratio was measured.It would be helpful to outline these regions in the images or, as mentioned above, use antibodies to define them.9. Figure 2C does not have indication of statistical significance for the comparisons stated in the text.Potential explanations for the different roles of Dhd and TrxT in long-term tumour development should be explored in the discussion.Related to this, does the analysis of the RNA-seq data from TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1 animals reveal why they have similar effect on mbt tumour development but do not synergistically contribute to long-term growth?10.Authors should clarify if there is any overlap between the affected M-tSDS and F-tSDS in the TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1 conditions.Would the limited overlap suggest that TrxT and dhd act in parallel rather than synergistically?This might also explain the differential effects on long-term tumour development.Additionally, the stronger effect observed in Df(1)J5 animals may be due to TrxT and dhd functional redundancy.Currently, there is limited evidence to suggest that TrxT and dhd act synergistically to regulate mbt tumour growth based on the presented data.
11. Authors should include a Venn diagram depicting affected genes (M-tSDS and F-tSDS) in the TrxTKO; l(3)mbtts1, dhdKO; l(3)mbtts1 and Df(1)J5; l(3)mbtts1 genotypes as this could clarify the percentage of overlap of gene signatures in these different conditions.Related to this point, authors could provide results from GO analysis to investigate whether specific functional clusters are altered in the different conditions.
12. In Figure 3E, authors should indicate more explicitly in the figure panel and/or figure legend which genes display significant differences in expression in the different samples.
13.In Figure S2C-F it is not clear if the graphs represent data from all tissues or data from male and female tissues separately, as shown in Figure 4.
14. Are TrxT and dhd also deregulated in other tumour types?Or is this specific for mbt tumours?This information could be provided to enhance the scope of the manuscript.
15. Authors conclude that TrxT and dhd cooperate in controlling gene expression between wild-type and tumour samples and that they act synergistically in the regulation of sex-linked gene expression in male tumour tissue.However, the link between the two observations (if indeed there is a link) has not been well explained.Is the effect on gene expression in tumours simply a result of the regulation of sexlinked transcription?

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The current manuscript provides additional information regarding the regulation of mbt tumours and establishes TrxT and Dhd as potential cancer-germline genes in Drosophila.This will be of interest to researchers studying basic mechanisms of tumourigenesis and could potentially lead to identification of genes that could serve as biomarkers of disease.However, for this, a more general role of TrxT and Dhd needs to be established, as well as their potential conserved role as cancer-germline genes needs to be established.**Summary:** -In this manuscript, the authors address the role of the thioredoxins Dhd and TrxT in the development and growth of mbt tumors, a sexually dimorphic brain tumor that derives from the expansion of the neuroephitelium.To this end, the authors have successfully generated dhd and TrxT knock-out mutants using CRISPR-Cas9 and show that both dhd and TrxT individual knock-out partially reduces the mbt tumorassociated brain phenotype.Moreover, using Df(1)J5, a deficiency that affects both TrxT and dhd, the recovery of the phenotype is enhanced.However, although concomitant expression of dhd and TrxT is required for proper tumor development, they show that only TrxT is necessary for the growth of allografts derived from male l(3)mbt tumors.This is interesting, not only because TrxT and dhd are never coexpressed in physiological conditions, but also because this data suggests that the pathways leading to l(3)mbt tumor development are different from the ones that contribute to tumor proliferation and aggressiveness.Moreover, the authors show that TrxT and dhd contribute to the emergence of the mbt tumour signature (MBTS) and sex-dimorphic signature (SDS) of tumours by analysing transcriptomic data of TrxT KO; l(3)mbt, dhd KO; l(3)mbt and Df(1)J5; l(3)mbt.In fact, through hierarchical clustering, the authors show that male Df(1)J5; l(3)mbt brain transcriptomic profile becomes closer to wild-type brains than l(3)mbt ts1 tumors.

How much time do
-This study presents novelty to the cancer research field and both the model and methodology used were appropriate.Nonetheless, this study deals with mbt tumors which are sexually dimorphic, as well as male and female germline-specific genes that in a tumor can alter male and female sex-dimorphic signatures, making this study very easy to become confusing to non-experts in the field if not written in a very clear way.Therefore, the text, especially in the results and discussion section, could be revised in general to improve the comprehension and flow of the manuscript, given that some sentences and paragraphs are hard to follow.In particular, the results section could benefit with more contextualization and a more detailed explanation of experiments.Moreover, the study is lacking some quantifications and a few additional experiments.These issues can certainly be addressed by reviewing the text as well as reorganizing and including a few quantifications and experiments as described below.I am an expert in Drosophila brain development and tumorigenesis.**Comments:** -In the first section of the results, as a first step to study the role of TrxT and dhd genes on mbt tumors the authors generate CRISPR knock outs of these genes and correctly validate them.However, afterwards, the experiment where the authors test the KO of these genes in a wild-type larva brain is not contextualized with the rest of the section.It might be best to first address the role of these genes in a tumor context and only then complement with the experiments in wild-type (in supplementary material).Fig 2 B and C -Indeed, the results suggest that TrxT seems to be responsible for most tumor lethality upon l(3)mbt allografts, but not dhd.This is curious since l(3)mbt; dhd KO brain tumors have the same partial phenotype as l(3)mbt; TrxT KO (fig 1A).It would be interesting to further explore these phenotypes by staining l(3)mbt; TrxT KO and l(3)mbt; dhd KO brains with, for instance, PH3 to understand if the number of dividing cells of these tumors could be different.In addition, to back up this information, the authors could look at what happens to l(3)mbt tumors with TrxT KO and dhd KO at a later stage of development (or to larva or pupa lethality if that is the case) and compare it with l(3)mbt brains.
-Fig 2 B -What happens to the medulla in a l(3)mbt brain tumor?Although the ratio of NE/BL is the same for wild-type and D(1)J5; l(3)mbt, it still seems that the medulla in D(1)J5; l(3)mbt brains is substantially bigger, although quantifications would be required.Do the authors know if the NE in D(1)J5; l(3)mbt brains is either proliferating less or differentiating more?-Figure S1 -Although the effects of TrxT KO and dhd KO in male mbt tumors seem to be enhanced in relation to female tumors, the authors should include some form of tumor quantification for female tumors like in Fig 2 A. Moreover in the 2nd section of the results, relative to Fig 1S in "...Df(1)J5; l(3)mbtts1 female larvae although given the much less severe phenotype of female mbt tumours, the effect caused by Df(1)J5 is quantitatively minor." to say "quantitatively" minor, the authors should include not only quantifications, but a form of comparison between female tumors vs. male tumors.
-Fig 3D -The hierarchical clustering was done according to which parameters?A brief explanation could help a better interpretation of this results section.
-Fig 3D -It could be beneficial for the authors to include an analysis of the downregulated genes shared between TrxT KO mbt tumors and dhd KO mbt tumors, as well as the genes that are not shared (besides MBTS genes).Could be something like a Venn diagram.
-Results section 3 -"Expression of nanos is also significantly down-regulated upon TrxT loss, but remains unaffected by loss of dhd" -to corroborate the idea that TrxT and dhd work as a pair, but contribute to different functions within the tumor, it would be interesting for the authors to do an allograft experiment of dhd KO; l(3)mbt male tissue with nanos knock down in the brain, if genetically possible.**Minor comments:** -In the first section of the results, the authors claim that "Consistent with the reported phenotypes of Df(1)J5...", but then the study is not mentioned.
-Fig 1 B -It is a bit confusing to follow where TrxT and dhd are in the Genome browser view.I am guessing we should follow the TrxT-dhd locus from A, but the authors could make it clearer.
-In the same section, in the next sentence, the homozygous and hemizygous is a bit confusing."...homozygous TrxTKO females, dhdKO males, and TrxTKO males", should be corrected.
-In the same section (Fig 1C ): "RNA-seq data also shows that TrxT is significantly upregulated in l(3)mbtts1 males compared to females (FC=7.06;FDR=1.10E-44) while dhd is not (FC=1.89;FDR=2.00E-14)."-But dhd is nevertheless upregulated, although less, in l3mbt males, right?The authors might need to rephrase.Moreover, these results will be easier to interpret if you add a male symbol in this image and a female symbol in Figure S1, otherwise, it might seem like the same figure if one does not properly read the legend.
-In results, section 2, "Consequently, in spite of the strong sex dimorphism of mbt tumours, the phenotype of Df(1)J5; l(3)mbtts1 larval brains is not sexually dimorph"to back this up, quantifications of Df(1)J5; l(3)mbtts1 female vs male tumor size, as well as statistical analysis are needed, like previously said.
-Last paragraph of section 2 of results -very long and confusing sentence.Please rephrase text.
-On section 3 of results: "The vas, piwi and CG15930 transcripts are not significantly down-regulated following either TrxT or dhd depletion alone."-in Fig 3E, not only these transcripts seem to suffer a slight downregulation, but there is also no statistical analysis supporting this.
-First paragraph of section 3 results -the first sentence is written in a confusing way.Moreover, more context is needed in the sentence afterwards: "we first focused on transcripts that are up-regulated in male mbt tumour samples compared to male wildtype larval brains (mMBTS)."but using which data?The RNA seq data?-Brief conclusion missing on the second paragraph of the last section of results.
-In the end of 3rd paragraph of last section of results: "...M-tSDS and F-tSDS genes is partially reduced in l(3)mbtts1 brains lacking either TrxT or dhd, but it is completely suppressed upon the lack of both."-"completely" might not be a correct word to use in this case, as there is still some small differences.
-4th paragraph of last section of results: Either mention the male results and then female (to be in order with the figure, as the female graphs come after the male graphs) or change the order in the figure.Also, this paragraph is not very clear, could benefit from a better explanation of the results and conclusions.
-Fig 4 C,D,E,F: to make it more clear, please write the name of the genotypes in question in the figure.

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This study presents an interesting new concept for Drosophila tumors, the cancer germline genes, which to my knowledge has been a poorly explored field, although it has a lot of potential.It is particularly interesting since it addresses the role of two germline specific thioredoxins, that are dispensable for somatic cells, but have a critical role in somatic mbt tumors, exploring new tumor vulnerabilities.This manuscript will benefit researchers in the field of cancer biology, in particular, to better understand cancer-testis (CT) genes and how they promote tumorigenesis, since the biological function for the most part remains unclear.

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We very much appreciate the reviewers' thorough comments and are sincerely grateful for their kind remarks on the novelty and interest of our manuscript.We are confident to have addressed all the points that they have raised including new data, as well as revised figures and text.

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All the revisions have been already carried out and included in the transferred manuscript.

> The number of the replicates/animals for the experiments described in Figures 1 and 2 should be reported either in the figure legends or in the methods (statistical analysis).
We have added the required numbers to the corresponding revised figures, as requested.

> A relevant part of the discussion repeats what the authors have already said in the results. I would recommend to reorganize this section, emphasizing the importance of these results in the context of human brain tumors.
Following our own style, we have written a very short (46 lines in length!) Discussion.We dedicate a few lines to highlighting two points: (1) the suggestion, derived from our allograft experiments, that the initial stages of tumour development and long-term tumour growth may be molecularly distinct events, and (2), the unique effect of the combined loss of TrxT and dhd on mbt tumour transcriptomics -unique because none of the suppressors of mbt reported before are as effective in erasing both the MBTS and SDS mbt signatures.Neither of these points are raised in Results.In the remaining few lines we put our results in the context of human Cancer/Testis and elaborate on the fact that the TrxT and dhd pair qualify as head-to-head, CT-X genes, like those reported in human oncology.This is as far as we are willing to go at this stage at emphasizing the importance of our results in the context of human tumours.

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Reviewer #2 > 1. Figures should include information regarding the sex of the larvae, particularly as there has been a previously reported sex-linked effect in the phenotypes analysed.(e.g. in Figure 2 and Figure S1, where Indication of the sex of the animals should be provided in the figure OK and not just in the figure legend).
We fully agree.Sex must always be taken into account as a biological variable.All the experiments reported in the manuscript were carried out with sexed samples, and were annotated accordingly in the original text.In compliance with the reviewer's request we have added this information also to the revised figure.
> 2. Data regarding fertility.Can this be shown in a table format?Are dhdKO females fully sterile?What are the fertility levels of Df(1)J5?
Please note that we are not discovering anything here but merely corroborating what has been published before: the lack of TrxT does not affect fertility in either sex; the lack of Dhd results in female sterility (Torres-Campana et al., 2022, Tirmarche et al., 2016, Svensson et al., 2003, Pellicena-Palle et al., 1997).Adding a table would not be justified.Moreover, it would be a rather simple table: all single-pair mating tests (n=10 for each genotype) with Trxt KO and Dhd KO males, and TrxT KO females were as fertile as control flies, while all single-pair mating tests (n=10) with Dhd KO females were sterile.> 3. Are dhd and TrxT the only genes affected by Df(1)J5?Is there transcriptional data from Df(1)J5 animals to suggest that nearby genes are not affected by the deficiency?Of particular interest would be to assess if snf is affected or not as it is a known regulator of gene expression and splicing.
Yes dhd and TrxT are the only genes affected by Df(1)J5.That is the case according to Flybase (citing Svensson et al., 2003, andSalz et al., 1994)   > 5. Related to Figure 1D.Additional neural markers could be assessed in dhdKO and TrxTKO flies.Whilst the gross morphology of the brain does not seem to be affected, there is a possibility that cell specification is affected.Specific markers for the NE, MED and CB could be used to assess this in more detail, particularly as the DE-cad images shown for dhdKO and TrxTKO flies seem to differ slightly from the control.
We believe that there may be a small misunderstanding here.We have made this point clear in the revised version by referring to substantial published data showing that expression of these two genes is restricted to the germline and that, female fertility aside, TrxT and dhd deficient flies' development and life span are perfectly normal.
If anything, Figure 1D is redundant.However, we would rather keep it as a control that our CRISPR KO mutants behave as expected.

> 7 cont. Related to this, in addition to the anti-DE-cadherin data, it would be informative to include immunofluorescence data using antibodies such as anti-Dachshund (lamina), anti-Elav (medulla cortex) and anti-Prospero (central brain and boundary between central brain and medulla cortex) (as assessed in e.g. Zhou and Luo, J Neurosci 2013) in the mbt tumour situation to accurately describe regions disrupted by the tumours.
There is no denying that taking advantage of the many cell-type specific markers that are readily available in Drosophila could be of interest.The same applies to cell cycle markers like PH3, FUCCI, and many others.However, we believe that interesting as they may be, none of these markers will give us the clue on the molecular basis of TrxT and Dhd tumour function that is, of course, the open burning question that we are trying to address now.> 8. Authors should clarify how the NE was defined when mbt tumours are generated, as it is severely affected.From the images provided, it is unclear which region corresponds to NE or how the NE/BL ratio was measured.It would be helpful to outline these regions in the images or, as mentioned above, use antibodies to define them.The requested statistical significance data for these comparisons were stated in the second last paragraph of that section.To make these data more prominent we have also added this information to revised Figure 2C.
> 9 cont.Related to this, does the analysis of the RNA-seq data from TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1 animals reveal why they have similar effect on mbt tumour development but do not synergistically contribute to longterm growth?
Unfortunately our analysis of the RNA-seq data from TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1 animals does not give us any clue that could help us understand why they have similar effect on mbt tumour development, but not in long-term growth (allografts).To further explore this point, we have added new Figure S3 that includes a Venn diagram showing the overlap between the affected mMBTS genes in TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1, together with the lists of enriched GOs among overlapping and non-overlapping genes.GO differences are

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tantalising, indeed, However, they do not immediately suggest any direct explanation for the different roles of Dhd and TrxT in long-term tumour development.
> 10.Authors should clarify if there is any overlap between the affected M-tSDS and F-tSDS in the TrxTKO; l(3)mbtts1 and dhdKO; l(3)mbtts1 conditions.Would the limited overlap suggest that TrxT and dhd act in parallel rather than synergistically?This might also explain the differential effects on long-term tumour development.Additionally, the stronger effect observed in Df(1)J5 animals may be due to TrxT and dhd functional redundancy.Currently, there is limited evidence to suggest that TrxT and dhd act synergistically to regulate mbt tumour growth based on the presented data.

See below.
> 11.Authors should include a Venn diagram depicting affected genes (M-tSDS and F-tSDS) in the TrxTKO; l(3)mbtts1, dhdKO; l(3)mbtts1 and Df(1)J5; l(3)mbtts1 genotypes as this could clarify the percentage of overlap of gene signatures in these different conditions.Related to this point, authors could provide results from GO analysis to investigate whether specific functional clusters are altered in the different conditions.
We have taken the liberty of fusing points 10 and 11 that are conceptually similar.The requested Venn diagrammes showing the overlap between the affected M-tSDS and F-tSDS genes in the TrxTKO; l(3)mbtts1, dhdKO; l(3)mbtts1, and Df(1)J5; l(3)mbtts1 conditions, and GO analysis are now shown in new Figure S5.
Unfortunately, these new data do not suggest any obvious explanation for the differential effects of these two genes, nor do they allow us to derive any further conclusions regarding the nature of the pathways through which TrxT and dhd cooperate to sustain mbt tumour growth.However, our analyses demonstrate that efficient suppression of mbt phenotypic traits (in larval brains) and transcriptome requires the combined elimination of both germline thioredoxins, while the effect of individual removal of either of them is only partial.These data demonstrate the synergistic nature of TrxT and dhd function in mbt tumour growth.
> 12.In Figure 3E, authors should indicate more explicitly in the figure panel and/or figure legend which genes display significant differences in expression in the different samples.
We apologise for not having made this point clear in the original version: All (21) genes shown in this Table are significantly downregulated in DfJ5;ts1 vs ts1.From these, nanos and Ocho are also significantly downregulated in TrxTKO;ts1 vs ts1, and Ocho, HP1D3csd, hlk, fj, Lcp9, CG43394, and CG14968 are significantly downregulated in dhdKO;ts1 vs ts1.These data have been included in the revised figure legend.Data on all other comparisons are included in Table S1.
> 13.In Figure S2C-F it is not clear if the graphs represent data from all tissues or data from male and female tissues separately, as shown in Figure 4.
Apologies for the confusion.All samples were from male tissues as indicated in the original figure legend.To make it more clear, we have labelled all four panels in the revised figure .> 14.Are TrxT and dhd also deregulated in other tumour types?Or is this specific for mbt tumours?This information could be provided to enhance the scope of the manuscript.

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Thank you for raising this point.TrxT and dhd are not dysregulated in the other tumour types that were analysed in Janic et al., 2010 (i.e pros, mira, brat, lgl and pins).
> 15.Authors conclude that TrxT and dhd cooperate in controlling gene expression between wild-type and tumour samples and that they act synergistically in the regulation of sex-linked gene expression in male tumour tissue.However, the link between the two observations (if indeed there is a link) has not been well explained.Is the effect on gene expression in tumours simply a result of the regulation of sex-linked transcription?
Our data show that TrxT and dhd synergistically contribute to the emergence of both the MBTS (i.e tumour versus wild type) and SDS (i.e.male tumour versus female tumour).The only certainty at this time regarding the interconnection between both signatures is that they overlap, but only partially, which answers one the questions raised by the reviewer: the effect on gene expression in tumours is not simply a result of the regulation of sex-linked transcription.Beyond that, the link (if indeed there is a link) between these two signatures has not been investigated.
The lack of insight on this issue is not surprising taking into account that, in contrast to classical tumour signatures (tumour versus healthy tissue), the concept of sex-linked tumour signatures is relatively new and only a handful of such signatures have been published.Moreover, the vast majority of classical tumour signatures have not been worked out in a sex-dependent manner.

Comments:
> -In the first section of the results, as a first step to study the role of TrxT and dhd genes on mbt tumors the authors generate CRISPR knock outs of these genes and correctly validate them.However, afterwards, the experiment where the authors test the KO of these genes in a wild-type larva brain is not contextualized with the rest of the section.It might be best to first address the role of these genes in a tumor context and only then complement with the experiments in wild-type (in supplementary material).
We do appreciate the reviewer's view, but respectfully disagree.In our opinion, the manuscript flows better by presenting the tools that we have generated in Figure 1, By corroborating published data showing that these two germline genes do not affect soma development (Torres-Campana et al., 2022, Tirmarche et al., 2016, Svensson et al., 2003, Pellicena-Palle et al., 1997) this first figure not only validates our CRISPR KO mutants, but also sets the stage to highlight their significant effect on a somatic tumour like mbt.

> -Fig 2 B -To back up the quantifications in Fig 2A the authors could include images of l(3)mbt ts1 tumors with TrxT KO and dhd KO also.
The requested images are shown in new figure Figure S2B.

> Fig 2 B and C -Indeed, the results suggest that TrxT seems to be responsible for most tumor lethality upon l(3)mbt allografts, but not dhd. This is curious since l(3)mbt; dhd KO brain tumors have the same partial phenotype as l(3)mbt;
TrxT KO (fig 1A).It would be interesting to further explore these phenotypes by staining l(3)mbt; TrxT KO and l(3)mbt; dhd KO brains with, for instance, PH3 to understand if the number of dividing cells of these tumors could be different.

In addition, to back up this information, the authors could look at what happens to l(3)mbt tumors with TrxT KO and dhd KO at a later stage of development (or to larva or pupa lethality if that is the case) and compare it with l(3)mbt brains.
We did explore the possibility of looking at later stages.Unfortunately, the onset of the lethality phase compounded by major tissue reshaping from larval to adult brain make these stages unsuitable to reach any meaningful conclusion.With regards to staining for PH3, we think that like FUCCI and a long list of other useful labels that could be explored, it is potentially interesting, but hardly likely to give us the clue on the molecular basis of TrxT and Dhd tumour function, that is of course the one important question that we are addressing now.

> -Fig 2 B -What happens to the medulla in a l(3)mbt brain tumor? Although the ratio of NE/BL is the same for wildtype and D(1)J5; l(3)mbt, it still seems that the medulla in D(1)J5; l(3)mbt brains is substantially bigger, although quantifications would be required. Do the authors know if the NE in D(1)J5; l(3)mbt brains is either proliferating less or differentiating more?
There are no significant differences in medulla/BL nor in CB/BL ratios.The corresponding quantifications have been added to the revised version.As for the question on proliferation versus differentiation, the simple answer is that we do not know.
-Figure S1 -Although the effects of TrxT KO and dhd KO in male mbt tumors seem to be enhanced in relation to female tumors, the authors should include some form of tumor quantification for female tumors like in Fig 2 A.
We have carried out the requested quantifications and added the results in a new panel in revised Figure S1A.

> Moreover in the 2nd section of the results, relative to Fig 1S in "...Df(1)J5; l(3)mbtts1 female larvae although given the much less severe phenotype of female mbt tumours, the effect caused by Df(1)J5 is quantitatively minor." to say "quantitatively" minor, the authors should include not only quantifications, but a form of comparison between female tumors vs. male tumors.
The requested quantification was published in Molnar et al., 2019.However, we agree on the convenience of doing it again with our new samples.The new data, that confirm published results, are now shown as a new panel in revised Figure S1C.

> -Fig 3D -The hierarchical clustering was done according to which parameters? A brief explanation could help a better interpretation of this results section.
The requested information has been added to the Methods section.Hierarchical clustering was done using the function heatmap.2 in R to generates a plot in which samples (columns) are clustered (dendogram); genes (rows) are scaled by "rows"; distance = Euclidean; and hclust method = complete linkage.Expression levels are reported as Row Z-score.

> -Fig 3D -It could be beneficial for the authors to include an analysis of the downregulated genes shared between
TrxT KO mbt tumors and dhd KO mbt tumors, as well as the genes that are not shared (besides MBTS genes).Could be something like a Venn diagram.

Full Revision
Thanks for pointing this out.New Figure S3 shows the requested Venn diagram, as well as the list of enriched GOs for each group.There are no enriched GOs in the list of overlapping genes.TrxTKO; l(3)mbtts1-specific genes are enriched for GOs related to game generation, sexual reproduction, germ cell development and simlar GOs.dhdKO; l(3)mbtts1 -specific genes are enriched for GOs related to chitin, molting and cuticle development.
Tantalising as they are, these observations do not immediately suggest any direct explanation for the different roles of Dhd and TrxT in long-term tumour development.We are happy to add this supplemental information, but we do not deem it worth of any further discussion at this point.

> -Results section 3 -"Expression of nanos is also significantly down-regulated upon TrxT loss, but remains unaffected by loss of dhd" -to corroborate the idea that TrxT and dhd work as a pair, but contribute to different functions within the tumor, it would be interesting for the authors to do an allograft experiment of dhd KO; l(3)mbt male tissue with nanos knock down in the brain, if genetically possible.
The suggested experiment is published.The gene in question (nanos) is a suppressor of mbt tumour growth: In a nanos knock down background, l(3)mbt allografts do not grow (Janic 2010).

Minor comments:
> -In the first section of the results, the authors claim that "Consistent with the reported phenotypes of Df(1)J5...", but then the study is not mentioned.

> -Fig 1 B -It is a bit confusing to follow where TrxT and dhd are in the Genome browser view. I am guessing we should follow the TrxT-dhd locus from A, but the authors could make it clearer.
Figure 1 has been changed to make this point more clear.
> -In the same section, in the next sentence, the homozygous and hemizygous is a bit confusing."...homozygous TrxTKO females, dhdKO males, and TrxTKO males", should be corrected.
We appreciate the suggestion, but would rather stick to classical terminology and refer to KO/KO females as homozygous and to KO/Y males as hemizygous.
We refer to comparing males versus females, not wild type versus tumours.The text has been rephrased in the revised version to make this point clear.

> -Fig 2 A (quantifications), should be after the confocal images (Fig 2 B).
We respectfully disagree on this minor point.We initially organised this figure in the order recommended by the reviewer, but we eventually found it easier to write the article using the order shown in the submitted figure.We would rather stick to this version.

> -Fig 2 B and Fig S1 -Please include an outline of at least neuroepithelia and, if possible, Central brain or medulla so
that these regions can more clearly identified.Moreover, these results will be easier to interpret if you add a male symbol in this image and a female symbol in Figure S1, otherwise, it might seem like the same figure Outlines and symbols have been added to the revised figure, as required.
> -In results, section 2, "Consequently, in spite of the strong sex dimorphism of mbt tumours, the phenotype of Df(1)J5; l(3)mbtts1 larval brains is not sexually dimorph" -to back this up, quantifications of Df(1)J5; l(3)mbtts1 female vs male tumor size, as well as statistical analysis are needed, like previously said.
The requested the new data is now shown in revised Figure S1C.
Thanks for pointing this out.We did of course generate the requested statistical analysis data, but failed to include it in the manuscript.Chi-square statistical test gives a p value=0.2346.These data have been added to the revised version.

> -Last paragraph of section 2 of results -very long and confusing sentence. Please rephrase text.
We have rephrased this sentence to make it shorter and clearer.

> -On section 3 of results: "The vas, piwi and CG15930 transcripts are not significantly down-regulated following either TrxT or dhd depletion alone." -in Fig 3E, not only these transcripts seem to suffer a slight downregulation, but there is also no statistical analysis supporting this.
There seems to be a misunderstanding here.The requested statistical data for each gene were shown in Table S1 > -First paragraph of section 3 results -the first sentence is written in a confusing way.Moreover, more context is needed in the sentence afterwards: "we first focused on transcripts that are up-regulated in male mbt tumour samples compared to male wild-type larval brains (mMBTS)."but using which data?The RNA seq data?Agreed; this paragraph has been amended in the revised version.
-Brief conclusion missing on the second paragraph of the last section of results.
As far as the results presented in this paragraph are concerned, we can only mention the two potentially interesting observations, which were pointed out in the original version: (i) the suggestion that nanos upregulation could be critical for sustained mbt tumour growth upon allograft, and (ii) the fact that three genes (vas, piwi and CG15930), also known to be required for mbt tumour growth, are downregulated in Df(1)J5; l(3)mbtts1, but remain unaffected following either TrxT or dhd depletion alone.We are unable to derive any other conclusion from these observations.

Full Revision
> -In the end of 3rd paragraph of last section of results: "...M-tSDS and F-tSDS genes is partially reduced in l(3)mbtts1 brains lacking either TrxT or dhd, but it is completely suppressed upon the lack of both."-"completely" might not be a correct word to use in this case, as there is still some small differences As requested, we have changed "completely" for "strongly".
> -4th paragraph of last section of results: Either mention the male results and then female (to be in order with the figure, as the female graphs come after the male graphs) or change the order in the figure.Also, this paragraph is not very clear, could benefit from a better explanation of the results and conclusions.
Point taken.Figure 4 has been changed and female graphs come before male graphs.The paragraph is clearer now.The conclusion from this paragraph is included in the final paragraph of this section.4 C,D,E,F: to make it more clear, please write the name of the genotypes in question in the figure .At the reviewer's request, the genotypes in question are now written in each panel.Please note that we did not do so before because all four panels correspond to the same genotype: Df(J5); l(3)mbtts1 vs l(3)mbtts1, as we mentioned in the original figure legend.1st Revision -Editorial Decision Dear Prof. Gonzalez, Thank you for the submission of your revised manuscript to our editorial offices.Before we can proceed with formal acceptance, I have these few editorial requests I ask you to address in a final revised manuscript:

> -Fig
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Fig 2 B -To back up the quantifications in Fig 2A the authors could include images of l(3)mbt ts1 tumors with TrxT KO and dhd KO also.

-
Fig 2 A (quantifications), should be after the confocal images (Fig 2 B).-Fig 2 B and Fig S1 -Please include an outline of at least neuroepithelia and, if possible, Central brain or medulla so that these regions can more clearly identified.
and confirmed by our own RNAseq data.No other transcripts, including snf, are affected by Df(1)J5.
figure legend.

> 7 .
Related to Figure 2B and Figure S1, three rows of images are presented for each genotype.It is unclear whether these correspond to brain lobes from different larvae or different confocal planes from the same animal.This should be clarified in the figure and/or figure legend.This point has been clarified as requested in the revised figure legend.Each group of three rows correspond to brain lobes from different larvae of the same genotype.
The figure has been modified to include the requested outlines defining the NE that indeed is correspond to the channel showing DE-Cadh staining.> 9. Figure2Cdoes not have indication of statistical significance for the comparisons stated in the text.Potential explanations for the different roles of Dhd and TrxT in long-term tumour development should be explored in the discussion.
Rev_Com_number: RC-2023-02273 New_manu_number: EMBOR-2024-59127V2 Corr_author: Gonzalez Title: TrxT and dhd are dispensable for Drosophila brain development but essential for l(3)mbt brain tumour growth 2nd Authors' Response to ReviewersAll editorial and formatting issues were resolved by the authors.pleased to accept your manuscript for publication in the next available issue of EMBO reports.Thank you for your contribution to our journal.

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